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v)/30% CH3CN + TFA (0.1% v/v)). The identity of the product was confirmed by
analytical HPLC with additional co-injection of EMPA reference standard. The
average time required for the synthesis from end of cyclotron bombardment to
end of synthesis was 34–36 min. The average radiochemical yield was 1.5–2.5%
(decay-uncorrected to trapped [11C]CH3I; n = 3). Chemical and radiochemical
purities were P95% with a specific activity range from 100 20 mCi/lmol
(EOB).
14. logD Determination: An aliquot (ꢀ50
l
L) of the formulated [11C]EMPA was
added to a test tube containing 2.5 mL of octanol and 2.5 mL of phosphate
buffer solution (pH 7.4). The test tube was mixed by vortex for 2 min and then
centrifuged for 2 min to fully separate the aqueous and organic phase. A
sample taken from the octanol layer (0.1 mL) and the aqueous layer (1.0 mL)
was saved for radioactivity measurement. An additional aliquot of the octanol
layer (2.0 mL) was carefully transferred to a new test tube containing 0.5 mL of
octanol and 2.5 mL of phosphate buffer solution (pH 7.4). The previous
procedure (vortex mixing, centrifugation, sampling, and transfer to the next
test tube) was repeated until six sets of aliquot samples had been prepared.
The radioactivity of each sample was measured in
a WIZARD Automatic
Gamma Counter (Perkin–Elmer, Waltham, MA).The logD of each set of samples
was derived by the following equation: logD = log(decay-corrected
radioactivity in octanol sample  10/decay-corrected radioactivity in
phosphate buffer sample).
15. Plasma protein binding assay: An aliquot of [11C]EMPA in saline (10
lL) was
added to a sample of baboon plasma (0.8 mL, pooled from two separate
animals). The mixture was gently mixed by repeated inversion and incubated
for 10 min at room temperature. Following incubation a small sample (20 lL)
was removed to determine the total radioactivity in the plasma sample (AT;
AT = Abound + Aunbound). An additional 0.2 mL of the plasma sample was placed in
the upper compartment of a centrifree tube (Amicon, Inc., Beverly, MA) and
then centrifuged for 10 min. The upper part of the centrifree tube was
discarded, and an aliquot (20 lL) from the bottom part of the tube was
removed to determine the amount of radioactivity that passed through the
membrane (Aunbound). Plasma protein binding was derived by the following
equation: %unbound = Aunbound  100/AT.
11.. EMPA: 1H-NMR (500 MHz, CDCl3): d 8.44 (d, J = 4.5 Hz, 1H), 8.35 (s, 1H), 7.94
(d, J = 2.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.55 (m, 1H), 7.38 (m, 2H), 7.17 (m,
3H), 6.58 (d, J = 8.5 Hz, 1H), 4.53 (s, 2H), 4.46 (s, 2H), 3.84 (s, 3H), 3.27 (q, 2H),
2.44 (s, 3H), 1.12 (t, J1 = J2 = 7.0 Hz, 3H). 13C-NMR (125 MHz, CDCl3): d 167.0,
163.3, 149.2, 148.9, 147.8, 140.0, 138.2, 136.5, 135.5, 133.1, 132.8, 132.6,
130.4, 129.7, 126.0, 123.4, 110.8, 53.6, 52.5, 46.2, 41.5, 20.6, 13.8. LC-MS calcd
for C23H26N4O4S (M): 454.2; Found (M+1): 455.4.
16. Rodent PET/CT acquisition: Male Sprague–Dawley rats were utilized in pairs,
anesthetized with inhalational isoflurane (Forane) at 3% in a carrier of 2 L/min
medical oxygen and maintained at 2% isoflurane for the duration of the scan.
The rats were arranged head-to-head in a Triumph Trimodality PET/CT/SPECT
scanner (Gamma Medica, Northridge, CA) featuring
a PET resolution of
approximately 1 mm. Rats were injected with standard references or vehicle
via a lateral tail vein catheterization at the start of PET acquisition. Dynamic
PET acquisition lasted for 60–90 min and was followed by computed
tomography (CT) for anatomic coregistration.
17. Baboon MR-PET acquisition: A female Papio Anubis baboon, deprived of food for
12 h prior to the study, was administered intramuscular ketamine (10 mg/kg)
and intubated. For maintenance of anesthesia throughout the study, the
baboon was provided 1–4% isoflurane (Forane) in a mixture of medical oxygen
and nitrogen. The baboon was catheterized antecubitally for radiotracer
injection and a radial arterial line was placed for metabolite analysis. MR-
PET images were acquired in a Biograph mMR scanner (Siemens, Munich,
Germany), with a PET resolution of 5 mm and field of view of 59.4 and 25.8 cm
(transaxial and axial, respectively). Dynamic PET image acquisition was
12. Precursor 1: 1H NMR (500 MHz, DMSO-d6): d 8.73 (d, J = 8.5 Hz, 2H), 8.11 (d,
J = 7.0 Hz, 1H), 7.83 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.56 (m, 1H), 7.37 (m, 2H),
7.28 (m, 2H), 6.21 (d, J = 10 Hz, 1H), 4.69 (s, 2H), 4.63 (s, 2H), 3.36 (q, 2H), 2.51
(s, 3H), 1.10 (t, J1 = J2 = 7.0 Hz, 3H). 13C NMR (125 MHz, DMSO-d6): d 168.3,
161.7, 143.3, 143.1, 142.1, 138.2, 137.7, 137.4, 133.7, 133.2, 130.3, 126.9, 126.8,
126.3, 119.6, 119.5, 119.0, 52.5, 46.1, 42.2, 20.6, 14.3. LC–MS calcd for
initiated followed by administration of the radiotracer in
a homogenous
solution of 10% ethanol and 90% isotonic saline. An MEMPRAGE sequence
began after 30 min of the baseline scan for anatomic coregistration. 4–5 mCi of
C
22H24N4O4S expected (M): 440.2; found (M+1): 441.3.
[
11C] EMPA was administered to the baboon for each scan.
13. 11CO2 was obtained via the 14N (p, 11C reaction on nitrogen with 2.5%
a
)
18. Image reconstruction and analysis: Dynamic data from the PET scans were
recorded in list mode and corrected for attenuation. Baboon data were
reconstructed using a 3D-OSEM method resulting in a full width at half-
maximum resolution of 4 mm. Rat PET data were reconstructed using a 3D-
MLEM method resulting in a full width at half-maximum resolution of 1 mm.
Reconstructed images were exported from the scanner in DICOM format along
with an anatomic CT for rodent studies and MRI for baboon scans. These files
were imported to PMOD (PMOD Technologies, Ltd) and manually coregistered
using six degrees of freedom. Volumes of interest (VOIs) were drawn manually
as spheres in brain regions guided by high resolution structural images (MRI
for baboon and CT for rats) and summed PET data, with a radius no less than
double that of the PET voxel size to minimize partial volume effects (4 mm for
baboon and 1 mm for rodent scans). A common VOI mask was applied to both
baboon scans. Time–activity curves (TACs) were exported in terms of decay
corrected activity per unit volume at specified time points with gradually
increasing intervals. The TACs were expressed as percent injected dose per unit
volume for analysis.
oxygen, with 11 MeV protons (Siemens Eclipse cyclotron), and trapped on
molecular sieves in a TRACERlab FX-MeI synthesizer (General Electric). 11CH4
was obtained by the reduction of 11CO2 in the presence of Ni-hydrogen at
350 °C and passed through an oven containing I2 to produce 11CH3I via a radical
reaction. 11CH3I was trapped in a TRACERlab FX-M synthesizer reactor (General
Electric) preloaded with
(6.0 mg) in dry DMSO (300
a
l
solution of precursor (3) (1.0 mg) and Cs2CO3
L) that had stirred at rt for 5 min prior to trapping.
The solution was stirred at rt for 3 min and water (1.2 mL) was added. The
reaction mixture was purified by reverse phase semi-preparative HPLC
(Phenomenex Gemini NX-C18, 250 Â 10 mm,
5 lm, 5.0 mL/min, 70%
H2O + TFA (0.1% v/v)/30% CH3CN + TFA (0.1% v/v)) and the desired fraction
was collected. The final product was reformulated by loading onto a solid-
phase exchange (SPE) C-18 cartridge rinsing with H2O (5 mL), eluting with
EtOH (1 mL), and diluting with saline (0.9%, 9 mL). The chemical and
radiochemical purity of the final product was tested by analytical HPLC
(Agilent Eclipse XDB-C18, 150 Â 4.6 mm, 1.0 mL/min, 70% H2O + TFA (0.1% v/